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Mitochondrial ribosomal small subunit (MRPS) MRPS23 protein–protein interaction reveals phosphorylation by CDK11-p58 affecting cell proliferation and knockdown of MRPS23 sensitizes breast cancer cells to CDK1 inhibitors

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Abstract

Background

Post-translational modification of some mitoribosomal proteins has been found to regulate their functions. MRPS23 has been reported to be overexpressed in various cancers and has been predicted to be involved in increased cell proliferation. Furthermore, MRPS23 is a driver of luminal subtype breast cancer. However, its exact role and function in cancer remains unknown.

Methods and results

Our previous study identified protein–protein interactions involving MRPS23 and CDK11A. In this study, we confirmed the interaction of MRPS23 with the p110 and p58 isoforms of CDK11A. Phosphoprotein enrichment studies and in vitro kinase assay using CDK11A/cyclin D3 followed by MALDI-ToF/ToF analysis confirmed the phosphorylation of MRPS23 at N-terminal serine 11 residue. Breast cancer cells expressing the MRPS23 (S11G) mutant showed increased cell proliferation, increased expression of PI3-AKT pathway proteins [p-AKT (Ser47), p-AKT (Thr308), p-PDK (Ser241) and p-GSK-3β (Ser9)] and increased antiapoptotic pathway protein expression [Bcl-2, Bcl-xL, p-Bcl2 (Ser70) and MCL-1] when compared with the MRPS23 (S11A) mutant-overexpressing cells. This finding indicated the role of MRPS23 phosphorylation in the proliferation and survival of breast cancer cells. The correlation of inconsistent MRPS23 phosphoserine 11 protein expression with CDK11A in the breast cancer cells suggested phosphorylation by other kinases. In vitro kinase assay showed that CDK1 kinase also phosphorylated MRPS23 and that inhibition using CDK1 inhibitors lowered phospho-MRPS23 (Ser11) levels. Additionally, modulating the expression of MRPS23 altered the sensitivity of the cells to CDK1 inhibitors.

Conclusion

In conclusion, phosphorylation of MRPS23 by mitotic kinases might potentially be involved in the proliferation of breast cancer cells. Furthermore, MRPS23 can be targeted for sensitizing the breast cancer cells to CDK1 inhibitors.

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Abbreviations

AKT:

Protein kinase B

BCL2:

B-cell lymphoma 2

CDK1:

Cyclind dependent kinase 1

CDK11A:

Cyclin dependent kinase–11A

CDK1i:

Indolyl methylene-2-indolinone anti-proliferative agent

COX2:

Cytochrome oxidase 2

c-Raf:

RAF proto-oncogene serine/threonine-protein kinase

DAP3:

Death associated protein -3

DMEM:

Dulbecco’s modified eagle medium

ECL:

Enhanced chemiluminescence

ELISA:

Enzyme-linked immunosorbent assay

FBS:

Fetal bovine serum

FP:

Flavopiridol

GAPDH:

Glyceraldehyde- 3-phosphate dehydrogenase

GSK-3β:

Glycogen synthase kinase 3 beta

GST:

Glutathione-S-transferase

IP:

Immuneprecipitation

KLH:

Keyhole limpet hemocyanin

LC/MS:

Liquid chromatography/mass spectrometry

MALDI:

Matrix-assisted laser desorption/ionization

MCL-1:

Myeloid cell leukemia 1

MRPS:

Mitochondrial ribosomal small subunit

MRPS23:

Mitochondrial ribosomal protein S23

p21:

CDK-interacting protein 1

PDK1:

Protein 3-phosphoinositide-dependent protein kinase-1

p-MRPS23 (Ser 11):

Phosphor-serine11 MRPS23 antobody

PPI:

Protein–protein interaction

PTEN:

Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase

PTM:

Post translational modification

S11A:

Ser 11 to Ala substituition mutant of MRPS23

S11G:

Ser 11>glutamin acid substitutiton mutant of MRPS23

ser:

Serine

TMB:

3,3′,5,5′-Tetramethylbenzidine

TOF:

Time-of-flight

WT:

Wild type

δTM:

Deletion mutant

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Funding

We acknowledge DST- Science and Engineering Research Board (SERB) for financial support. R. P. Oviya is supported by the Senior Research Fellowship (SRF-Direct) from Council of Scientific and Industrial Research (CSIR).

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Authors and Affiliations

  1. Department of Molecular Oncology, Cancer Institute (WIA), Adyar, Chennai, Tamil Nadu, 600020, India

    Revathi Paramasivam Oviya, Krishna Priya Thangaretnam, Balaji Ramachandran, Priya Ramanathan, Subramani Jayavelu, Gopisetty Gopal & Thangarajan Rajkumar

  2. Department of Molecular Oncology, Cancer Institute (WIA), Chennai, 600036, India

    Gopisetty Gopal

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  1. Revathi Paramasivam Oviya
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Contributions

GG and RPO planned and performed the experiments, TR, GG, and RPO analyzed the data, and wrote the manuscript. SJ assisted in MALDI and nLC/MS/MS experiments in the proteomics facility of our department, PR performed the flow cytometry experiment; BR performed the animal experiments. TR, assessed the patient clinical data for the study.

Corresponding author

Correspondence to Gopisetty Gopal.

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The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

This study is not a clinical trial and does not involve human subjects. All the tissues used for research purpose was obtained following informed consent, from Tumor bank, Department of Oncopathology, Cancer Institute (WIA). Animal studies were carried out with prior approval from the Institutional Animal Ethics Committee (IAEC) of Cancer Institute (W.I.A), Adyar, Chennai, India with IAEC approval number: CIWIA0119WR. Care of animals complied according to CPCSEA (Committee for the purpose of Control and Supervision of Experiments on Animals) guidelines, Government of India.

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Oviya, R.P., Thangaretnam, K.P., Ramachandran, B. et al. Mitochondrial ribosomal small subunit (MRPS) MRPS23 protein–protein interaction reveals phosphorylation by CDK11-p58 affecting cell proliferation and knockdown of MRPS23 sensitizes breast cancer cells to CDK1 inhibitors. Mol Biol Rep 49, 9521–9534 (2022). https://doi.org/10.1007/s11033-022-07842-y

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